Histoplasmosis is a poorly understood disease, in part because of the wide spectrum of syndromes that are possible following infection by Histoplasma capsulatum. The organism may remain confined to the lungs or may disseminate to other tissues; the infection may resolve with little symptomatology, may cause life-threatening illness, or may reactivate many years after all signs and symptoms have vanished. Some of this variation in pathogenesis clearly relates to the immune status of the host, and these aspects are being studied by a number of other research groups. In contrast, the efforts of this laboratory are centered on understanding how variations and adaptations by the organism itself could influence the manifestation of disease. The overall objective of this grant renewal remains unchanged from the original application: to study characteristics that define H. capsulatum as a pathogen. An emphasis has been placed on employing a variety of in vitro models of histoplasmosis as primary tools in studying pathogen-host interactions. A consistent theme (unique to this laboratory) is the use of a wide variety of geographically diverse and genetically distinct H. capsulatum strains, allowing broad-based comparisons of characteristics potentially related to virulence. In general, the planned research program is designed to exchange observations about H. capsulatum between molecular biology systems and cell biology systems. This application for 4 years of renewed support focuses on the following three research goals: I. To continue development of molecular genetic systems for cloning and gene disruption in H. capsulatum. The proposed work will take advantage of this group's recently published DNA transformation methodology for H. capsulatum. URA5 will continue to serve as a selectable marker for reverse genetics strategies and in a telomere-based shuttle vector. II. To refine our macrophage and epithelial cell systems as in vitro models for H. capsulatum infection and persistence. Macrophage-like cell lines, primary macrophage cultures, and respiratory epithelial cells will be used for studying H. capsulatum-host cell interactions, especially the effects on intracellular pH and calcium levels. III. To continue studying (using the systems described above)the genetic basis and mechanisms of action of virulence-associated phenotypes. Molecular biology will play a key role in evaluating the function and importance of CBP, a calcium-binding protein produced by H. capsulatum. Additional studies are proposed to characterize the ability of H. capsulatum to modulate phagolysosomal pH in non-activated and activated macrophages.